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Investigations on the anti-inflammatory and anti-allergic activities of the leaves of Uncaria guianensis (Aublet) J. F. Gmelin

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Uncaria guianensis (Aublet) J. F. Gmelin is an herbal medicine from tropical areas of South and Central America. We investigated the anti-inflammatory and anti-allergic properties of an ethanolic extract of U. guianensis leaves, containing alkaloids, flavonoids and phenol carboxylic acids, as revealed by thin layer chromatography (TLC). Oral pre-treatment with U. guianensis inhibited zymosan-induced paw oedema (500 mg/paw) and pleural exudation (100 mg/kg) within 4 h (25-200 mg/kg). U. guianensis (100 mg/kg) inhibited total leukocyte and neutrophil numbers in the pleural cavity 4 h after zymosan stimulation. Pre-treatment with U. guianensis (100 mg/kg, p.o.) inhibited total leukocyte, neutrophil and eosinophil recruitment into the pleural cavity 24 h after LPS (250 ng/cavity, i.t.). Pre-treatment with U. guianensis inhibited paw oedema (25-200 mg/kg) induced by ovalbumin (OVA) within 1 h, and neutrophil and eosinophil recruitment into the mice pleural cavity 24 h after OVA (100 mg/kg). In vitro data revealed that U. guianensis impaired LPS-induced nitric oxide and CXCL8 generation by murine peritoneal macrophages, as well as OVA-induced interleukin-5 synthesis by previously sensitized spleen cells. These results demonstrate that U. guianensis leaves provide effective anti-inflammatory and anti-allergic activities.
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Inammopharmacology 14 (2006) 48–56
0925-4692/06/020048-9
DOI 10.1007/s10787-006-1509-5
© Birkhäuser Verlag, Basel, 2006
Inammopharmacology
Research Article
Investigations on the anti-inammatory and anti-allergic
activities of the leaves of Uncaria guianensis (Aublet) J. F. Gmelin
M. V. Carvalho
1
, C. Penido
1
, A. C. Siani
2
, L. M. M. Valente
3
and M. G. M. O. Henriques
1, *
1
Departamento de Farmacologia Aplicada, Far-Manguinhos, Fundação Oswaldo Cruz, Rua Sizenando Nabuco 100, 21041-250, Rio de Janeiro, RJ,
Brazil, Fax: ++5521 25642559, e-mail: gracahen@far.ocruz.br
2
Departamento de Produtos Naturais, Far-Manguinhos, Fundação Oswaldo Cruz, Rua Sizenando Nabuco 100, 21041-250, Rio de Janeiro, RJ, Brazil
3
Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, Bloco A, 21910-240,
Rio de Janeiro, RJ, Brazil
Received 18 October 2005; revised 10 January 2006; accepted 11 January 2006
Abstract. Uncaria guianensis (Aublet) J. F. Gmelin is an
herbal medicine from tropical areas of South and Central
America. We investigated the anti-inammatory and anti-
allergic properties of an ethanolic extract of U. guianensis
leaves, containing alkaloids, avonoids and phenol carboxy-
lic acids, as revealed by thin layer chromatography (TLC).
Oral pre-treatment with U. guianensis inhibited zymosan-in-
duced paw oedema (500 mg/paw) and pleural exudation (100
mg/kg) within 4 h (25–200 mg/kg). U. guianensis (100 mg/
kg) inhibited total leukocyte and neutrophil numbers in the
pleural cavity 4 h after zymosan stimulation. Pre-treatment
with U. guianensis (100 mg/kg, p.o.) inhibited total leuko-
cyte, neutrophil and eosinophil recruitment into the pleural
cavity 24 h after LPS (250 ng/cavity, i.t.). Pre-treatment
with U. guianensis inhibited paw oedema (25–200 mg/kg)
induced by ovalbumin (OVA) within 1 h, and neutrophil and
eosinophil recruitment into the mice pleural cavity 24 h after
OVA (100 mg/kg). In vitro data revealed that U. guianensis
impaired LPS-induced nitric oxide and CXCL8 generation
by murine peritoneal macrophages, as well as OVA-induced
interleukin-5 synthesis by previously sensitized spleen cells.
These results demonstrate that U. guianensis leaves provide
effective anti-inammatory and anti-allergic activities.
Key words: Uncaria guianensis; Anti-inammatory; Anti-
allergic; Pleurisy, Paw oedema; Nitric Oxide; Cytokines
Introduction
Uncaria guianensis (Aublet) J. F. Gmelin and U. tomentosa
(Willd.) DC. are closely related species of the Rubiaceae
family, popularly known as cat’s claw (unha-de-gato or uña
de gato in South America). Both species are large woody
vines occurring at the Amazon rain forest and other tropical
areas of South and Central America, and are interchange-
ably used in traditional medicine, since they share the
same ethnomedical applications (reviewed by Heitzman
et al., 2005). The decoction of their barks is used for the
treatment of diabetes, cancer, intestinal affections (Vilches,
1997; Laus & Keplinger, 2003) and inammatory condi-
tions such as arthritis (reviewed by Heitzman et al., 2005).
The dried and powered leaves of U. guianensis are used to
treat wounds and abscesses (Jones, 1995). The chemical
characterization of such species revealed so far alkaloids
(whole plant), avonols (bark) and triterpene glycosides
(bark) (Laus & Keplinger, 2003; Sandoval et al., 2002;
Yépez et al., 1991).
Despite the broad use of U. guianensis in traditional
medicine, very few studies have assessed its therapeutic
properties. Previous studies using a decoction of the barks
supported its anti-inammatory effects in vitro through dif-
ferent mechanisms. Sandoval and coworkers (2002) showed
that water extract of the barks of U. guianensis was able to
inhibit TNF-a mRNA and protein synthesis in stimulated
RAW cells in vitro, nitrite formation and apoptosis. Moreo-
ver, the anti-oxidant activity of the barks of U. guianensis
has also been demonstrated (Sandoval et al., 2002; Piscoya et
al., 2001). A clinical study has demonstrated the efcacy of
U. guianensis to treat patients with osteoarthritis of the knee,
a phenomenon associated to its ability to inhibit TNF-a and
PGE
2
synthesis (Piscoya et al., 2001).
In the present study we have assessed the anti-inamma-
tory activity of the ethanolic extract of U. guianensis leaves,
using murine models of pleurisy and paw oedema. We also in-
vestigated the ability of such extract to inhibit the allergic re-
sponse to antigenic challenge in previously sensitized mice.
* Corresponding author
Vol. 14, 2006 Pharmacological effects of Uncaria guianensis 49
was estimated as the difference of volume (µL) between the stimulated
and non-stimulated paws. Allergic paw oedema was induced in animals
previously sensitized with a subcutaneous injection of 50 µg of ovalbumin
(OVA) plus 5 mg of Al(OH)
3
, in a nal volume of 100 µL. Fourteen days
later, oedema was triggered by an i.pl. stimulation with OVA (3 µg/paw).
Pleurisy
Pleurisy was induced as described by Henriques and coworkers (1990).
Briey, an adapted needle (13 × 5 gauge) was inserted into the right side
of the thoracic cavity to enable the intrathoracic (i.t.) administration of zy-
mosan (100 µg/cavity) or LPS (250 ng/cavity), diluted in sterile saline to a
nal volume of 100 µl per cavity. Allergic pleurisy was induced by an i.t.
injection of OVA (12.5 µg/cavity) in 14 days previously sensitized mice
by a subcutaneous (s.c.) injection (100 µl) of an emulsion of complete
Freund’s adjuvant containing 200 µg of OVA). Control groups received an
i.t. injection of the same volume of vehicle. At different time points after
stimulation, mice were killed by inhalation of carbon dioxide and their
thoracic cavities were washed with 1 ml of PBS containing heparin (20
IU/mL). Total leukocyte counts were performed in an automated particle
counter (Z1; Coulter-California). Differential leukocyte counts were de-
termined in cytocentrifuged smears stained with May-Grünwald-Giemsa
dye using an oil immersion objective (100 X). For quantication of pro-
tein extravasation in the pleural cavity, animals received Evans blue dye
(25 mg/kg) intravenously (i.v.) 24h before stimulation. Pleural washes
were centrifuged (1,155 g, 10 min) to remove cells, and the Evans blue
in the supernatant was quantied by measuring the absorbance at 600 nm
using a plate reader (Spectramax 190, Molecular Devices, CA). Results
are expressed as µg of Evans blue dye per cavity.
Cell Viability Assay
The effect of U. guianensis extract on the viability of C57Bl/6 perito-
neal macrophages was determined by MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) assay (Mosmann, T., 1983). The
yellow tetrazolim salt MTT is cleaved by the mitochondrial activity of
viable cells, forming a purple formazan derivative. Cells (2.5 × 10
5
/well)
were incubated with U. guianensis extract (0.5, 5, 50 and 500 µg/mL)
for 24 h at 37 °C, and then DMSO (dimethyl sulfoxide) was added to
dissolve formazan crystals. Analysis was performed at 540 nm in a mi-
croplate reader (Molecular Devices).
Nitric Oxide Production
Peritoneal macrophages recovered from C57Bl/6 mice were ressuspend-
ed in complete RPMI (RPMI 1640 supplemented with 10% fetal bovine
serum and gentamicin 25 µg/mL). Macrophage viability was determined
by trypan blue exclusion. Cells were plated onto 96-well at culture
plates (2.5 × 10
5
cells per well) and allowed to adhere for 1 h. Adherent
cells (98% macrophages) were incubated with different concentrations
of U. guianensis (0.5, 5, 50 and 500 µg/mL) in a nal volume of 200 µL
per well, in the presence or in the absense of LPS (30 ng/mL), at 37 °C/
5% CO
2
. Dexamethasone (100 µg/mL) was used as reference inhibitor.
Twenty-four h later, the amount of NO
2,
was evaluated in the supernatant
by the Griess method (Green et al., 1982). Equal volume of the culture
supernatant treatment groups and Griess reagent (1% sulphanilamide and
0.1% N-[naphthyl] ethylenediamine; 1:1) were mixed and the absorb-
ance was measured on a microplate reader at the wavelength of 540 nm.
The amount of nitrite was calculated from a NaNO
2
standard curve.
Enzyme-linked immunosorbent assay
Levels of IL-5 were analysed in vitro in the supernatant of splenocytes
recovered from previously sensitized (50 mg OVA/5 mg Al(OH), s.c.)
Materials and Methods
Plant material and preparation of extracts
The leaves of U. guianensis were collected in Juruena, Mato Grosso,
Brazil. A voucher specimen was deposited in the Central Herbarium
of Universidade Federal do Mato Grosso (UFMT), Brazil, under the
number 24,715, identied by the botanist Pierro Delprete (New York
Botanic Garden). The leaves were air-dried at room temperature and
milled in a blender. The extract was prepared by exhaustive extraction of
the dried and powered leaves (30.0 g) with ethanol at room temperature,
followed by ltration and evaporation of the solvent at low pressure
yielding 3.30 g of the dried extract (11%).
Thin layer chromatography proles of the extract
In order to obtain preliminary information of the chemical constituents
of the leaves extract, the oxindole alkaloid and polyphenol chromato-
graphic proles were made through thin layer chromatography (TLC).
The analyses were carried out by applying 10 µl of a methanol solution
(c=50 mg/ml) of the extract to pre-coated silica-gel 60F
254
plates (Mer-
ck), in two different systems. For the oxindole alkaloid prole was used
the method described by Valente et al., 2005 with hexane-ethyl acetate
5:95 as mobile phase and visualization of the spots by UV irradiation at
254 nm. The crude extract prole was compared at the same chromato-
graphic condition to a reference sample with the six current biomarker
pentacyclic oxindole alkaloids and to extracts of leaves and bark of U.
tomentosa. For the polyphenolic prole, chloroform-methanol-formic
acid 3:1:0.1 was used as mobile phase and visualization was done by
UV irradiation at 254 nm followed by spraying with NP/PEG reagent
and observation under UV light at 365 nm (Wagner & Bladt, 1996).
Animals
Male Swiss Webster (20–30 g) and C57Bl/6 mice (18–20g), from the
Oswaldo Cruz Foundation (Rio de Janeiro, Brazil) colony were lodged
in a room with controlled temperature (23 ± 2 °C) and lighting (lights
on from 7 a.m. – 7 p.m.), with free access to lab chow and tap water. All
experimental procedures were performed according to the Ethics Com-
mittee for Animal Care and Use (CEUA/FIOCRUZ, license 0050/00)
and to the ethical guidelines of International Association for the Study
of Pain (Zimmermann, 1983).
Treatments
Twelve hour-fasted animals were treated orally (p.o.) with 25, 50, 100
or 200 mg/kg of U. guianensis ethanolic extract diluted in ltered water,
1h before stimulation. In designed experiments, diclofenac (100 mg/kg
in ltered water, p.o., 1h prior) and promethazine (30 mg/kg in sterile
saline, intraperitoneally, i.p.) were administered 1 h prior stimulation. In
another set of experiments, dexamethasone (2 mg/Kg, i.p.) was used as
reference inhibitor, and administered 24 and 1 h before stimulus. In all
instances, the nal volume injected was 200 µL. Control groups received
the same volume of the corresponding vehicle alone.
Paw oedema
Paw oedema was induced as described by Henriques and co-workers
(1987). Briey, animals received an intraplantar (i.pl.) injection of 50
µL of zymosan (500 µg/paw) into one hind paw. The contralateral paw
was injected with the same volume of vehicle (sterile saline) and used as
control. The volumes of each hind paw were measured by using a digital
plethysmometer (Ugo Basile, Italy) 4h after stimulation. The oedema
50 M. V. Carvalho et al. Inammopharmacology
C57Bl/6 mice. Briey, spleens were pressed through a stainless steel
mesh, and suspended in RPMI 1640 medium. Red blood cells were
lysed by hypotonic shock. Erythrocyte-free spleen cells were then
washed and cell viability was determined by trypan blue exclusion.
Cells were added to a 24 well plate (5 × 10
6
cells/mL) in RPMI 1640
supplemented with 10% fetal bovine serum, incubated with U. guianen-
sis extract (0.5, 5, 50 and 500 µg/mL), or dexamethasone (100 µg/mL)
for 1 h at 37 °C at 5% CO
2
, and stimulated with OVA (10 µg/mL) for
6 h. Analysis was performed in the supernatant by sandwich enzyme-
linked immunosorbent assay (ELISA) using matched antibody pairs
from PharMingen (San Diego, CA), according to the manufacturer’s
instructions.
Material
Zymosan, lipopolysaccharide (LPS, from Escherichia coli serotype
055:B5), ovalbumin, dexamethasone, RPMI 1640, MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), gentamicin,
trypan blue, thioglycolate, Griess reagent (sulfanilamide and N-(1-
naphthyl, 10:1) ethylenediamine dihydrochloride, BSA (bovine serum
albumin), o-phenylenediamine dihydrochloride (OPD), red blood cell
lysing buffer and phosphate-buffered saline (PBS) were purchased from
Sigma Chemical Co. (St. Louis, MO). Promethazine hydrochloride (i.e.
Fenergan
) was obtained from Aventis (Brazil). Streptoavidin-con-
jugated horseradish peroxidase was purchased from Zymed (USA).
Aluminum hydroxide was obtained from Sano Synthelabo (USA).
Diclofenac potassium and saline solution were obtained from the Insti-
tute of Drug Technology – Farmanguinhos (Fiocruz, Brazil). Evans blue
dye was purchased from Merck (Germany). Sodium heparin (Liquem-
ine
®
) was obtained from Roche (Brazil). Ethanol (PA grade), used for
the extraction, dimethyl sulfoxide (DMSO) and H
2
SO
4
was purchased
from VETEC, Brazil. The following antibodies were purchased from
PharMingen (San Diego, CA): puried anti-murine IL-5 mAb, puri-
ed anti-murine TNF-a mAb, puried anti-murine CXCL1 (KC) mAb,
biotinilated anti-IL-5 mAb, biotinilated anti-TNF-a mAb, biotinilated
anti-CXCL8 mAb, as well as the respective murine recombinant IL-5,
TNF-a and CXCL-8 (KC).
Statistical Analysis
Data were reported as the mean ± S.E.M. and were analyzed statisti-
cally by means of analysis of variance (ANOVA) followed by Newman-
Keuls-Student test or Student t test. Values of p< 0.05 were regarded as
signicant.
Results
Alkaloid and polyphenol chromatographic prole of the
extract of the leaves of U. guianensis
The TLC alkaloid prole of the extract of the leaves of U.
guianensis seems to present speciophylline, uncarine F, pter-
opodine and isopteropodine along with other non-identied
alkaloids all at low concentration as judged by comparison
of the Rf value and the intensity of their spots with those
of the reference sample and also with those of the leaves
and bark of U. tomentosa (Valente et al., 2005). The TLC
polyphenolic prole of the plant extract showed the presence
of avonoids and phenol carboxylic acids by analyzing the
color of the spots after detection with NP/PEG-UV (Wagner
& Bladt, 1996).
Effect of the ethanolic extract of the leaves of U. guianensis
on zymosan-induced paw oedema and pleural exudation
The anti-inammatory properties of the U. guianensis leaf
were assessed by the zymosan-induced paw edema and
pleurisy in mice previously treated with U. guianensis ex-
tracts (25, 50, 100 and 200mg/kg, p.o.). As shown in gure
1, the i.pl. injection of zymosan induced a signicant paw
oedema in Swiss mice within 4 hours. Pre-treatment with
the anti-inammatory compound used as reference inhibi-
tor (diclofenac, 100 mg/kg, p.o.) was able to inhibit 45% of
oedema formation induced by zymosan stimulation. The oral
pre-treatment with U. guianensis extract signicantly inhib-
ited paw oedema triggered by zymosan at all doses tested
(25–200 mg/kg), with maximal inhibition of 31% achieved
with 100 mg/kg. Table 1 demonstrates that the i.t. injection
of zymosan (100 µg/cavity) induced a signicant protein
extravasation into the mice pleural cavity 4 h after stimu-
lation. Pre-treatment with diclofenac (100 mg/kg, p.o., 1 h
Fig. 1. Effect of the pre-treatment with U. guianensis ethanolic extract
(25–200 mg/kg, p.o., 1 h before stimulation) on paw edema induced by
the i.pl. injection of zymosan (500 µg/paw). Diclofenac (100 mg/kg,
p.o.) was used as reference inhibitor. Analysis was performed 4 h after
stimulation. Results are expressed as the mean ± SEM of at least 7 ani-
mals. Statistically signicant differences (p 0.05) between treated and
non-treated groups are indicated by an asterisk.
Table 1: Effect of ethanolic extract of U. guianensis upon zymosan-in-
duced plasma pleural protein extravasation
Groups Protein (µg/mL)
Saline 1.22 ± 0.21
Zymosan 3.29 ± 0.79
Diclofenac 1.07 ± 0.13 +
U. guianensis
1.39 ± 0.30 +
Results are expressed as mean ± EPM of three representative experi-
ments. p 0.05 compared to saline; + p 0.05 compared to zymosan
(ANOVA followed by Neman-Keuls test)
Vol. 14, 2006 Pharmacological effects of Uncaria guianensis 51
prior zymosan stimulation) and U. guianensis extract (100
mg/kg, p.o.) signicantly inhibited protein extravasation into
the pleural cavity (67% and 58%, respectively). Such result
supports the anti-edematogenic activity of U. guianensis eth-
anolic extract, observed in paw oedema.
Effect of the ethanolic extract of the leaves of U. guianensis
on zymosan-induced leukocyte accumulation
According to previous data, the i.t. injection of zymosan in-
duced a marked increase in total leukocyte numbers in mice
pleural cavity within 4 h after simulation (gure 2A), due to
neutrophil accumulation (gure 2B; Henriques et al., 1990;
Sampaio et al., 2000a). The oral administration of U. guian-
ensis extract (100 mg/kg, p.o.) signicantly diminished total
leukocyte numbers (44%), due to the inhibition of neutrophil
inux (57%; gures 2A, B) into the mice pleural cavity. The
reference anti-inammatory drug diclofenac (100 mg/kg,
p.o., 1 h prior) also inhibited zymosan-induced leukocyte in-
ux into the mice pleural cavity (66% and 78% of inhibition
of total leukocytes and neutrophils, respectively).
Effect of the ethanolic extract of the leaves of U. guianensis
on LPS-induced leukocyte recruitment
It has been demonstrated that the LPS i.t. stimulation does
not induce pleural protein extravazation, but induces an im-
portant leukocyte accumulation in the pleural cavity, due to
an early neutrophil inux, followed by eosinophil and mono-
nuclear cells accumulation (Bozza et al.,1994a; Penido et al.,
1997). In fact, LPS stimulation induced a signicant increase
in total leukocyte, neutrophil and eosinophil numbers in the
mice pleural cavity within 24 h (gure 3), a phenomenon
inhibited by dexamethasone (2 mg/kg, i.p., 24 and 1 h prior
LPS i.t. injection). We observed that the pre-treatment with
U. guianensis ethanolic extract (100 mg/kg, p.o.) also sig-
nicantly inhibited total leukocyte accumulation (33% of
inhibition; gure 3 A) due to an impairment of neutrophil
and eosinophil recruitment (50% and 41%; gures 3B, C,
respectively), conrming the anti-inammatory activity of
U. guianensis.
Effect of the ethanolic extract of the leaves of U. guianensis
on paw oedema induced by antigenic challenge
The anti-allergic property of U. guianensis was evaluated on
paw oedema triggered by the OVA. As illustrated in gure
4, the anti-histaminic compound prometazine was able to
inhibit signicantly (40%) the oedema formation induced by
antigenic challenge. The oral pre-treatment with the extract
also signicantly inhibited the oedema formation induced by
OVA, at all doses tested (25–200 mg/kg, p.o.), in a dose-de-
pendent manner (R=0.96), with maximal inhibition of 61%.
Such result suggests a potent anti-edematogenic and anti-al-
lergic activity of such extract.
Effect of the ethanolic extract of the leaves of U. guianensis
on leukocyte inux during allergic pleurisy
The murine model of allergic pleurisy was used to assess
the anti-allergic activity of U. guianensis leaf extract. Ac-
cording to previous published data, we observed an impor-
tant leukocyte accumulation in the mice pleural cavity 24
h after i.t. antigenic challenge, mainly due to eosinophil
and neutrophil inux (Xanthoudakis et al., 1996; Sampaio
et al., 2000b) (gure 5). The previous treatment with U.
guianensis (100 mg/kg, p.o.) markedly reduced the total
leukocyte (A), neutrophil (B) and eosinophil (C) migration
into pleural cavity. It is noteworthy that the inhibition of
neutrophil and eosinophil accumulation by U. guianensis
(81% for neutrophils and 77% for eosinophils, respectively)
was similar to the observed after dexamethasone pre-treat-
ment (84% for neutrophils and 80% for eosinophils, respec-
tively).
Fig. 2. Effect of the U. guianensis ethanolic extract (100 mg/kg, p.o.) on
zymosan (100 µg/cavity, i.t.)-induced total leukocyte (A) and neutrophil
recruitment (B) into the mice pleural cavity. Diclofenac (100 mg/kg,
p.o.) was used as reference inhibitor, and administered 1 h before zy-
mosan i.t. injection. Analysis was performed 4 h after stimulation. Re-
sults are expressed as the mean ± SEM of at least 7 animals. Statistically
signicant differences (p 0.05) between stimulated and non-stimulated
groups are indicated by an asterisk, whereas + indicates differences be-
tween treated and non-treated groups.
*
+
+
*
+
+
52 M. V. Carvalho et al. Inammopharmacology
Assessment of cell viability in mice peritoneal macrophages
In order to investigate the effect of U. guianensis upon the
production of inammatory mediators, in vitro models were
used. Therefore, we rst investigated the cytotoxic effects
of U. guianensis extract on C57Bl/6 mice peritoneal macro-
phages by MTT assay. As shown in table 2, the incubation
of macrophages with U. guianensis extract for 24 h was not
citotoxic at all concentrations analyzed (0.5–500 µg/mL).
Effect of the ethanolic extract of the leaves of U. guianensis
on LPS-induced nitric oxide, CXCL1 and TNF-a
production by mouse peritoneal macrophages
LPS stimulation (30ng/mL) was able to induce signicant
increase in CXCL8, TNF-a and NO generation by murine
peritoneal macrophages, as observed in gure 6. The pre-
treatment with U. guianensis extract (0.5–500 µg/mL) was
able to signicantly inhibit CXCL8 production, with maxi-
mal inhibition of 24%, at 0.5 µg/mL, in the same extent as
dexamethasone (20% at 100 µg/mL) (gure 6A). The pre-
treatment with such extract also inhibited NO production
(5–500 µg/ml), in a dose-dependent manner (R=0.96; with
maximal inhibition of 61%, at 500 µg/mL; gure 6C). By
contrast, U. guianensis extract failed to inhibit on TNF-a
production (gure 6B) by LPS-stimulated murine peritoneal
macrophages.
Fig. 3. Effect of U. guianensis ethanolic extract (100 mg/kg, p.o.) on
total leukocyte (A), neutrophil (B) and eosinophil (C) recruitment into
the mice pleural cavity induced by LPS (250 ng/cavity, i.t.). Dexametha-
sone (2 mg/kg, i.p.) was used as reference inhibitor, and administered 24
and 1 h before stimulation. Analysis was performed within 24 h. Results
are expressed as the mean ± SEM of at least 7 animals. Statistically
signicant differences (p 0.05) between stimulated and non-stimulated
groups are indicated by an asterisk, whereas + indicates differences be-
tween treated and non-treated groups.
Fig. 4. Effect of the pre-treatment with U. guianenses ethanolic extract
(25–200 mg/kg, p.o., 1 h before stimulation) on paw edema induced
by the i.pl. injection of OVA (3 µg/paw) in previously sensitized mice.
Promethazine (30 mg/kg, p.o.) was used as reference inhibitor. Analysis
was performed 1 h after stimulation. Results are expressed as the mean ±
SEM of at least 7 animals. Statistically signicant differences (p 0.05)
between treated and non-treated groups are indicated by an asterisk. The
dose-effect relationship between doses was detected by linear regres-
sion, and the correlation coefcient was R=0.96.
Table 2: Macrophages viability after incubation with ethanolic extract
of U. guianensis
Groups Viability (%)
Control 100%
U. guianensis (0.5µg/mL)
100%
U. guianensis (5µg/mL)
100%
U. guianensis (50µg/mL)
100%
U. guianensis (500µg/mL)
100%
Vol. 14, 2006 Pharmacological effects of Uncaria guianensis 53
Effect of the ethanolic extract of the leaves of U. guianensis
on IL-5 production by murine splenocytes
ELISA assay demonstrated increased levels of IL-5 in the
supernatant of OVA-stimulated splenocytes recovered from
previously sensitized C57Bl/6 mice (table 3). The pre-treat-
ment of cells with U. guianensis extract (0.5–50 µg/mL) was
able to inhibit on IL-5 production, with maximal inhibition
of 39%, at 50 µg/mL).
Discussion
To date, very few studies have assessed the therapeutic
properties of U. guianensis. The results of the present work
contribute to validate the ethnobotanical use of the leaves
of U. guianensis as anti-inammatory agent, and also re-
vealed its anti-allergic potential. The present results clearly
Fig. 5. Effect of the oral pre-treatment with U. guianensis ethanolic ex-
tract (100 mg/kg, 1 h prior stimulus) on total leukocyte (A), neutrophil
(B) and eosinophil (C) recruitment induced by OVA (12.5 µg/cavity, i.t.)
into the pleural cavity of previously sensitized mice. Dexamethasone (2
mg/kg, i.p.) was used as reference inhibitor, and administered 24 and 1
h before challenge. Analysis was performed within 24 h. Results are ex-
pressed as the mean ± SEM of at least 7 animals. Statistically signicant
differences (p 0.05) between stimulated and non-stimulated groups are
indicated by an asterisk, whereas + indicates differences between treated
and non-treated groups.
Fig. 6. Effect of the ethanolic extract of U. guianensis (0.5–500 µg/ml)
on CXCL1 (A), TNF-a (B) and nitric oxide (C) production by mouse
peritoneal macrophages stimulated with LPS (30 ng/ml). Cells were
pre-treated with the extract for 1 h and analysis were performed within
4 or 24 h. Results are expressed as mean ± SEM of two experiments
with three samples each. Statistically signicant differences (p 0.05)
between stimulated and non-stimulated groups are indicated by an as-
terisk, whereas + indicates differences between treated and non-treated
groups.
54 M. V. Carvalho et al. Inammopharmacology
demonstrate the anti-inammatory activity of the ethanolic
extract of U. guianensis leaves in different in vivo models.
The anti-oedematogenic properties of U. guianensis was
rst investigated, since vascular permeability is an important
feature of early inammatory reactions, leading to plasma
extravasation and consequent oedema formation at sites of
injury. The oral pre-treatment with U. guianensis extract
showed a marked anti-edematogenic activity, by inhibiting
the paw oedema and plasma exudation in the pleural cavity
of mice induced by zymosan. These data are similar to the
results obtained by Aguilar and colleagues (2002), with U.
tomentosa, which was also able to inhibit paw oedema in-
duced by carrageenan in mice.
The present report also shows, for the rst time, that U.
guianensis leaves presents an important anti-allergic activity,
since it was able to inhibit paw oedema induced by antigenic
challenge in previously sensitized mice. Although the pro-
duction of oedematogenic mediators was not directly inves-
tigated, the hypothesis that U. guianensis leaves is somehow
able to inhibit their effects, might be put forward. Since his-
tamine plays a major role in vascular permeability enhance-
ment during allergic responses, it is especially noteworthy
that the extent and potency of the inhibition provided by U.
guianensis extract (at 200 mg/kg) against OVA-induced paw
edema was similar to that of promethazine, the reference
anti-histaminic compound tested, suggesting the anti-hista-
minic property of U. guianensis. However, further investiga-
tion is necessary to elucidate the mechanisms by which U.
guianensis is exerting its anti-oedematogenic activity.
Leukocyte inux from blood into the inammatory site
represents a crucial step in inammatory responses. Early
inammatory reactions are usually characterized by the in-
ux of neutrophils into the inamed tissues, whereas at late
time points, an intense inux of eosinophils and mononu-
clear cells is usually observed. According to previous data,
our results showed a signicant neutrophil accumulation in
the pleural cavity 4 h after zymosan stimulation (Sampaio et
al., 2000a). Such accumulation was signicantly inhibited by
the U. guianensis extract. Cisneros et al. (2005) reported that
mice treated with U. tomentosa also showed reduced neu-
trophil numbers in broncoalveolar lavage (BAL) uid and
bronchioles of inamed lungs, suggesting that the inhibition
of neutrophil accumulation might be one of the important
mechanisms by which such species elicit its anti-inamma-
tory effects.
In accordance to previously published data, LPS and
OVA challenge induced a marked increase in total leukocyte
numbers in the pleural cavity within 24 h, due to an inux
of mononuclear cells, neutrophils and eosinophils (Bozza et
al., 1994a,b; Larangeira et al., 2001). U. guianensis extract
was also able to inhibit the inux of total leukocytes into
the pleural cavity triggered by LPS and OVA within 24 h,
due to the inhibition of neutrophils and eosinophils, with no
changes observed in mononuclear cell numbers (data not
shown). It is noteworthy that the marked effect of U. guian-
ensis extract pre-treatment in LPS and allergen-induced leu-
kocyte recruitment was also very close to the one induced by
dexamethasone.
No studies have previously reported the effects of U.
guianensis or U. tomentosa on the production of neutrophi-
lotactic mediators. The increased levels of the chemokine
CXCL1 in the inammatory site has been implicated in
the mobilization of neutrophils in different conditions and
experimental models, such as during the course of rheuma-
toid and experimental arthritis and allergic inammation
(López-Armada et al., 2002, Danahay et al., 1997). Here, we
show that the U. guianensis pre-treatment was able to inhibit
CXCL1 synthesis by murine macrophages stimulated with
LPS in vitro, suggesting it might be an important mechanism
by which U. guianensis impaires neutrophil inux in vivo.
In addition to that, U. guianensis extract also inhibited NO
generation, according to previous publish data (Sandoval-
Chacón et al., 1998), suggesting an additional mechanism
for the reduction of neutrophil inux (Farsky et al., 2004,
Salvemini et al, 1996).
It is plausible that the anti-allergic property of U. guian-
ensis, marked by its ability to inhibit eosinophil inux, be
due to a decreased synthesis of eosinophilotactic mediators.
It is known that interleukin (IL)-5 plays a central role in the
orchestration of eosinophil inltration, and that during al-
lergic responses IL-5 cooperates with eotaxin for eosinophil
migration in vivo and in vitro (Collins et al., 1995 and unpub-
lished data). We have observed an important increase in IL-5
synthesis by splenocytes after antigenic challenged in vitro.
Such phenomenon was inhibited by U. guianensis extract,
supporting the in vivo inhibition of allergic eosinophilia
in the mice pleural cavity. It has been demonstrated that
LPS-induced eosinophil accumulation depends on a neo-
synthesized protein not yet identied, but it occurs through
a mechanism independent of IL-5 or eotaxin (Bozza et al.,
1994a; 1994b), therefore, the mechanisms by which U.
guianensis inhibits LPS-induced eosinophil accumulation
has to be further investigated. It is interesting to note that U.
tomentosa extracts have been shown to inhibit NF-kB acti-
vation (Sandoval-Chacón et al., 1998), an activity that can
respond for various effects found in that species, since NF-
kB coordinates the expression of multiple genes that encode
inammatory cytokines and chemokines, including IL-5 and
eotaxin (Yang et al., 1998). However, no data have been pub-
lished concerning NF-kB inhibition by U. guianensis.
Pre-clinical studies on U. tomentosa extracts are much
more frequent and reported then studies on U. guianensis.
On the other hand, it has been previously demonstrated that
both cat’s claw species present a similar qualitative chemical
prole (Vilches, 1997) and that is probably the reason why
both species are reciprocally used for the same purposes (re-
Table 3: Effect of ethanolic extract of U. guianensis upon IL-5 produc-
tion by splenocytes stimulated with ovalbumin
Stimulus IL-5 (pg/mL)
Saline 0.0 ± 0.0
Ovalbumin 2.3 ± 20
Dexametasone 0.0 ± 0.0 +
U. guianensis (0.5µg/mL)
2.4 ± 1+
U. guianensis (5µg/mL)
1.7 ± 1+
U. guianensis (50µg/mL)
1.4 ± 1+
Results are expressed as mean ± EPM of three experiments. p 0.05
compared to saline; + p 0.05 compared to ovalbumin (ANOVA fol-
lowed by Neman-Keuls test)
Vol. 14, 2006 Pharmacological effects of Uncaria guianensis 55
viewed by Heitzman et al., 2005). Six pentacyclic oxindole
alkaloids, speciophylline, mitraphylline, pteropodine, un-
carine F, isopteropodine and isomitraphylline, are currently
considered to be their biochemical markers and are used to
standardize commercial herbal medicines. These alkaloids
are found in several parts of cat´s claw species, and are usu-
ally more abundant in U. tomentosa than in U. guianensis
(Laus & Keplinger, 2003). Sandoval et al. (2002) reported
that the aqueous extract of the barks of U. guianensis showed
a greater antioxidant and anti-inammatory activity than the
similar extract of U. tomentosa despite its lower alkaloid and
avanol content. However, comparison of the anti-inamma-
tory activity of the bark hydroalcoholic extract of U. tomen-
tosa against its bark aqueous extract revealed that the rst,
with higher alkaloid content, was signicantly more active
than the last one (Aguilar et al., 2002). The bioassay guided
fractionation of the anti-inammatory active extract of U. to-
mentosa led to the isolation of a triterpenoid acid glycoside
as a bioactive compound (Aquino et al., 1991). On the other
hand Gonçalves et al. (2005) provided some evidences for
an antioxidant mechanism underlying the anti-inammatory
activity of the aqueous extract of the barks of U. tomentosa
that could be correlated to polyphenol compounds present
in the extract. All these data point towards, as commented in
Heitzman et al. (2005), a possible synergic action of differ-
ent compounds in the anti-inammatory activity. However,
efforts are being done in order to elucidate the chemical
composition of the ethanolic U. guianensis extract used in
the present work.
In conclusion, the present results indicate that leaves of
U. guianensis presents a marked anti-inammatory activity,
supporting its use in traditional medicine. In addition, an im-
portant anti-allergic effect of U. guianensis is here described
for the rst time. Such effects rely on the inhibition of nitric
oxide, CXCL1 and IL-5 synthesis.
Acknowledgments. The present work was supported by grants from
CNPq and FIOCRUZ (Brazil). The authors are indebted to Andrea Sur-
rage for technical assistance, to Dr. Peter May and N.G.O. Pro-Natura
(Brazil) for the kind donation of the plant material and to Dr. Hélida B.
N. Borges from the Central Herbarium of the UFMT (Brazil).
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The jungles of Central and South America contain two predominant species of cat’s claw (Uña de Gato), Uncaria tomentosa (Willd. ex Schult.) DC. and Uncaria guianensis (Aubl.) J.F. Gmell, which are used in traditional medicine mainly for their anti-inflammatory properties. However, a wealth of compounds have been isolated from these two vines of the Rubiaceae family, including alkaloids, flavonoids and terpenoids, showing a wide range of activities: anti-inflammatory, anti-oxidative, hypotensor, antiviral, smooth muscle relaxant, antispasmodic, gastrointestinal mucosa protector, antiarrhythmic, anticonvulsant, analgesic, anti-leishmaniosis, cytostatic, cytotoxic, hypoglycaemizing, anticholestatic, antihistaminic, hepatoprotective, diuretic, antiulcer, immunostimulating and sedative effects. Some of these activities have been confirmed in both in vitro and in vivo models.
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1. The injection of 100 or 300 micrograms of carrageenin into the mouse paw or pleural cavity produced a delayed inflammatory reaction at 48 h while platelet activating factor (PAF)-induced paw oedema and pleurisy were maximal 30 min after its injection. 2. The PAF antagonist, WEB 2086, failed to inhibit mouse paw oedema and pleurisy induced by PAF, but reduced the first phase of oedema (1-4 h) induced by carrageenin without interfering with the second one (48-72 h). In contrast, another structurally-related PAF antagonist, WEB 2170, inhibited dose-dependently both oedema and pleurisy induced by PAF and by carrageenin (48 h). 3. Repeated injections of PAF into the mouse paw or pleural cavity led to significant autodesensitization. The animals desensitized to PAF and injected with carrageenin also displayed a significantly reduced oedema. 4. Our results suggest that PAF may be involved in the inflammatory response to carrageenin in mice. Furthermore, because the different receptor antagonists displayed distinct effects against PAF itself, different sites for in vivo interaction of PAF are available and are species- and drug-dependent.